보안과제 ( ), 일반과제 ( o )

Transcription

1 보안과제 ( ), 일반과제 ( o )

2

3 .. 1.,.,,. in-vitro in-vivo gel probiotics. (Dioscorea batatas) ( ) ( ) ( ), %,,,,.,,,,,,

4 ( ),.,..,,,,,.,. glucomannan, mucin, choline, arginine, ton. 70%. 10 a (1,000 m 2 ) 370, ,

5 , , , Disocorea batatas.,.,,,.,,,,,,,.,,, glucomannan,,..,,,,, formulation - 4 -

6 ..,., Nigeria stew.,,,., ) (1) (2) 2) (1) (2) 3) (1) (2) - 5 -

7 (3) (4) 2. 1) (1) (2) 2) (1) (2) 3) (1) (2) (3) (4) 3. (In vivo test) 1) - 2) - 3)

8 . 1.,,. 1% 60, 90 in-vitro, 8 newtrase protamex., protamex. Hexane, chloroform, ethylacetate, butanol ethanol, HMG-CoA reductase inhibition bile acid binding capacity ethylacetate ethanol, ethanol. Ethanol, 30,000 dalton HMG-CoA reductase inhibition bile acid binding capacity, gel filtration 29,000 dalton 16,000 dalton in-vitro, Lactobacillus acidophilus, Enterococcus flavescens, Enterococcus faecalis var. liquefaciens, Lactococcus lactis, Lactobacillus casei 5. Lactobacillus acidophilus., ethanol butanol, ethanol. Ethanol, 30,000~50,000 dalton gel chromatograpy 66,000 dalton

9 3...,,

10 . 1. : 1. Myung-Ki Lee, et al. Inhibition of Browning and Preference Improvements of Dioscorea batatas through the Addition of Sugar Alcohols and Organic Acids. J Food Sci Nutr. 14: (2009) 2. : : 9. 4 : : Mouse : Mouse

11 . 4 : : Lactobacillus acidophilus : : : : : 1 ( ) - : - :

12 SUMMARY. Title of Research Development of products using functional components and hematocele improvement effect from Dioscorea batatas. Objectives and Significance of Research 1. Objectives and significance of research For the aims to broaden the utilization of Dioscorea batatas, this study developed enzyme hydrolysate and fermented product manufacturing methods, and performed separation and purification of elements that potentially have functional effects on improvement blood stream. Furthermore, we developed diversity processed food with these functional elements to increase the Dioscorea batatas consumption. Products development was performed with raw materials that having functionality evidenced by in-vitro and in-vivo experiments. As a long-term market circulation product which can preserved for a long period, Dioscorea batatas chocolate was developed, and as a short-term market circulation products which can be easily prepared for substitute meal and have a probiotic benefits, beverage, pottage, and sweet jelly were developed. These applications on processed food promote consumption and commercialization of Dioscorea batatas. Dioscorea batatas, belongs to Dioscoreaceae family, is grown in mountain field and grassland which is hermaphrodites, has both male and female reproductive organs, proliferate themselves and occurring various heritable variations. Approximately, 600 species of Dioscoreaceae family have been reported. More than 80% of Dioscorea batatas domestically produced in Gyeongsang province, and therefore advanced studies of unique quality properties, preference, uniformity, stable preservation of Dioscorea batatas are required for promoting as a new indigenous crop. Dioscorea batatas is used in Asia as the herbal medicine, which is utilized after

13 processing such as uncooked or steamed-and-dried its periderm after peeling. There have been several reports of beneficial effects on anti-oxidative and glucose regulating, anti-cancer, obesity, immune-regulation, intestinal function, and lipid metabolism. Sinnongbonchokyung, Chinese medical book, says pharmacological effect of old Dioscorea batatas is placed after wild ginseng. Therefore, it seems to be a worthwhile subject to investigate biological functionality and nutritional superiority of Dioscorea batatas and develop high value food material as well as processed foods, in terms of academical, technical, and economical aspects. A. Technical aspects Dioscorea batatas is eaten raw, steamed and cooked, or grated Dioscorea batatas served with the york, fried and coated with syrup. Lately, its starch used as a energy source and healthy food in the US. Thus, it has become important to investigate particular active constituent for improving blood flow and develop a new food as a high valuable material. Variety elements such as glucomannan, one of mucous polysaccharides, mucin, choline, arginine in Dioscorea batatas have been reported have various bioactive benefits include lowering blood cholesterol. However, few studies have been conducted to support these functional superiorities scientifically. Therefore, there are a lot of studies required to establish technology for increasing effective use of Dioscorea batatas. B. Economic and industrial aspects Dioscorea batatas grows over the central-south region, and some species even grows in north area in Korea. Almost 4,311 tons of Dioscorea batatas produced in last year(2008), and half of them are from Andong in Gyeongbuk province of Korea. In terms of producing area, it is over 70% of total area in Korea. Farmers make 3.7 million won per 10 a (1,000 m 2 ) on average in last year, currently more than 920 farm households growing Dioscorea and sell after cleaning and cutting process. Beneficial effects of Dioscorea batatas became known to the world, more than 50 kinds of processed products such as powder, tea, and soup, are exported to the US and

14 Southeast Asia, which worth of more than 336,000 dollars, through Dioscorea Processing Plant in Andong Nonghyup. As well as, record 5 billion won in domestic sales rewards for their effort of product development and improvement of species of Dioscorea as a regional specialty. As above, economical value of Dioscorea is drastically rising that could revive local economy and increase export by effective utilization. 2. Current domestic and international technology A. Preliminary research a. Domestic preliminary research Dioscorea batatas is a perennial herbaceous plant belonging to Dioscoreaceae family grown over the country mountain. Most studies on Dioscorea batatas are performed with variety cultivated in fields, and few studies on functional aspects found in the literature. Some of main components contained in Dioscorea batatas include glucomannan, mucin, arginine, and choline, are known to effective for diabetes, pulmonary tuberculosis, disinfection, antidote, rheumatism. Scientific research, however, has not been sufficiently investigated. Thus, we attempt to purify the active components and establish the functional effects of Dioscorea batatas. We expecting effective extraction of bioactive compounds based on advanced information on how to separate functional component from ginseng, ballon flower, and lanceolata. b. International preliminary research In Japanese cuisine, Dioscorea is eaten raw and grated, after only a relatively minimal preparation. The raw vegetable is starchy and bland, mucilaginous when grated, and may be eaten plain as a side dish, added to noodle, or using for confectionary source. In China, fried Dioscorea eaten coated with sweet syrup. Dioscorea batatas is a main carbohydrate source for West African, particularly in Nigeria, it is a gourmet food served as hot stew. Recently in America, Dioscorea starch

15 is a good source of energy which is easily digest, and use as an ingredient in bread, biscuit, and soup, especially for children and the infirm. As described, Dioscorea batatas is consumed as a simply process food, without a consideration of the functionality on blood flow improvement. So, more efforts needed to develop products that can maximize the health benefits of Dioscorea batatas.. Scope and Contents of Research 1. Blood stream improvement effects of isolated functional material using hydrolysis enzymes from Dioscorea batatas. 2. Blood stream improvement effects of isolated functional material using lactic acid bacteria from Dioscorea batatas. 3. Development of processed foods using Dioscorea batatas and their functional materials. IV. Conclusion and recommendations 1. Blood stream improvement effects of isolated functional material using hydrolysis enzymes from Dioscorea batatas. To develop functional material from Dioscorea batatas, observed optimal concentration, temperature and time using 8 types of carbohydrate and protein hydrolysis enzyme. As a result, the optimal condition difference for 8 type of each enzyme, but mainly in the 1% of concentration, 60 and 90 min of enzyme reaction condition, especially protein enzymatic hydrolysates (newtrase, protamex) showed the most blood stream improvement effects. The result of animal experiments using enzymatic hydrolysates, protamex hydrolyzate had the highest blood cholesterol-lowering effect. So we conducted solvent separation using protamex hydrolyzate. Hexane, chloroform, ethylacetate, butanol and ethanol solvent fractionation carried out, and HMG-CoA reductase inhibition and bile acid binding capacity were measured. The ethylacetate fraction and ethanol fraction were higher their effects, through the animal experiments,

16 ethanol fraction was finally selected. The ultrafiltration of ethanol fraction, 30,000 dalton in size that HMG-CoA reductase inhibition and bile acid binding capacity were superior to, gel filtration through confirm the final result of molecular size less than 29,000 dalton and 16,000 dalton, respectively. 2. Blood stream improvement effects of isolated functional material using lactic acid bacteria from Dioscorea batatas. Fermented Dioscorea batatas used by 33 species of lactic acid bacteria and observed improvement of in-vitro blood flow effects, fermentation materials of Lactobacillus acidophilus, Enterococcus flavescens, Enterococcus faecalis var. liquefaciens, Lactococcus lactis, Lactobacillus casei had a good activity of HMG-CoA reductase inhibition and bile acid binding capacity, respectively. And animal experiment, the effect of cholesterol control in the Lactobacillus acidophilus fermented material was excellent, therefore we selected. For Separation of the active components from fermented material, we enforced solvent fractionation. And ethanol and butanol fractions were the best improvement in the blood flow effect and animal experiments were selected for the final fraction of ethanol fraction. Ethanol fraction was separated by ultrafiltration method, 30,000~50,000 dalton was excellent for the improvement in the blood flow effect. The final step, separated by gel chromatograpy, and substance were found to be less than 66,000 dalton. 3. Development of processed foods using Dioscorea batatas and their functional materials. For increase consumption promotion and expandation of processed food on Dioscorea batatas, we development various kinds of products. First, as a long-term storage capabilities in a convenient product, chocholate (using protamex hydrolysate) and sweet jelly (using lactic acid bacteria fermented material) prepared. The raw material of chocolate and jelly, can used not only functional components but the raw, powder and steamed Dioscorea batatas for take advantage of a wide variety of manufacturing methods and materials that were standardized basis. Second, for spread as a convenient

17 meals, prepared by fermented beverage and portage. Almost products had a good activity of control blood flow effect in the animal experiments. The serum cholesterol level showed similar with normal fat diet group. Although these products evaluated a good sensory properties

18 CONTENTS Chapter 1. Introduction 32 Chapter 2. Materials and Methods Development powder processing of Dioscorea batatas 35 A. Materials 35 B. General ingredients analysis of Dioscorea batatas 35 C. Change of ingredients in Dioscorea batatas by dry procedure 37 D. Effect of additives for antibrowning and improvement of sensory properties Determination optimal condition of functional components isolation from Dioscorea batatas hydrolysate 40 A. Manufacturing of Dioscorea batatas hydrolysate 40 B. Optimization for enzyme hydrolysis of Dioscorea batatas Determination optimal condition of functional components isolation of fermented Dioscorea batatas 43 A. Manufa cturing of fermented Dioscorea batatas 43 B. Preperation and optimization of fermented Dioscorea batatas Functional properties of enzyme hydrolysate and fermented product in selected optimum conditions 45 A. DPPH radical scavenging effect 45 B. Fibrinolytic effect 45 C. HMG-CoA reductase inhibition activity 46 D. Bile acid binding capacity Purification of functional components and stabilization on blood stream improvement

19 activity 49 A. Solvent fractionation of hydrolysate Dioscorea batatas 49 B. Solvent fractionation of fermented Dioscorea batatas 50 C. Ultrafiltration of solvent fraction 50 D. Gel chromatography 52 E. In-vitro investigation of blood stream improvement activity 52 F. Quantitative analysis of charbohydrates from components 52 G. Quantitative analysis of protein from components 53 H. Quantitative analysis of lipid from components 53 I. Molecular weight observation of components 54 K. Stabilization of functional fractions 6. Development of processed products using Dioscorea batatas 55 A. Manufacture of Dioscorea batatas wine 55 B. Development of long term storage products 57 1) Manufacture of Dioscorea batatas chocolate 57 2) Manufacture of Dioscorea batatas sweet jelly 62 C. Development of short term storage products 70 1) Manufacture of Dioscorea batatas fermented beverage 70 2) Manufacture of Dioscorea batatas pottage 76 D. Quality characteristics of products 81 E. Storage stability of processed products F. Research for application of developed functional food In-vivo experimentation of blood stream improvement activity 84 A. In-vivo experimentation of hydrolysate and fermented Dioscorea batatas 84 B. In-vivo experimentation of functional fraction from Dioscorea batatas 85 C. In-vivo experimentation of processed products 86 D. Determination of blood stream improvement activity

20 Chapter 3. Results and Discussion Development powder processing of Dioscorea batatas 93 A. General ingredients analysis of Dioscorea batatas 93 B. Change of ingredients in Dioscorea batatas by dry procedure 94 C. Effect of additives for antibrowning and improvement of sensory properties Determination optimal condition of functional components isolation from Dioscorea batatas hydrolysate 100 A. Effect of temperature and type on hydrolytic enzyme 100 B. Effect of concentration and type on hydrolytic enzyme 103 C. Effect of time on hydrolytic enzyme Determination optimal condition of functional components isolation of fermented Dioscorea batatas 111 A. ph, titratable acidity, total bacteria and lactic acid bacteria of fermented Dioscorea batatas 111 B. Sugar, glucose and reducing sugar content of fermented Dioscorea batatas Functional properties of enzyme hydrolysate and fermented product in selected optimum conditions 121 A. Fibrinolytic effect and HMG-CoA reductase inhibition effect of hydrolysate 121 B. Bile acid binding capacity of hydrolysate 123 C. Fibrinolytic effect of fermented Dioscorea batatas 125 D. HMG-CoA reductase inhibition effect of fermented Dioscorea batatas 126 E. Bile acid binding capacity of fermented Dioscorea batatas Purification of functional components and stabilization on blood stream improvement activity 131 A. Blood stream improvement activity of hydrolysate 131 B. Blood stream improvement activity of solvent fraction from hydrolysate

21 C. Blood stream improvement activity of fermented Dioscorea batatas 139 D. Blood stream improvement activity of solvent fraction from Dioscorea batatas 141 E. Blood stream improvement activity of ultrafiltration components 145 F. Quantitative analysis and blood stream improvement activity of gel chromatography components 145 G. Stabilization of functional fractions 6. Development of processed products using Dioscorea batatas 176 A. Physicochemical properties of Dioscorea batatas wine addition with rice 176 B. Physicochemical properties of Dioscorea batatas wine addition with oriental herbs 179 C. Quality properties of chocolate 181 D. Quality properties of sweet jelly 184 E. Quality properties of fermented beverage 190 F. Quality properties of pottage 197 G. Storage stability of processed products H. Research for application of developed functional food In-vivo experimentation of blood stream improvement activity 208 A. In-vivo experimentation of hydrolysate and fermented Dioscorea batatas 208 B. In-vivo experimentation of functional fraction from Dioscorea batatas 216 C. In-vivo experimentation of processed products 223 Chapter 4. Conclusion 238 Chapter 5. References

22 SUMMARY CONTENTS DPPH. (Fibrinolytic activity). HMG-CoA (3-hydroxy-methylglutaryl coenzyme A) reductase. (Bile acid binding capacity)

23 5.,.... gel chromatography.. Gel chromatography. Gel chromatography. Gel chromatography ) 2). 1) 2) in-vivo

24 ph,,., 4.. HMG-CoA reductase. (Bile acid binding capacity).. HMG-CoA reductase Gel chromatography

25 in-vivo

26 1 Dioscorea batatas Dioscorea japonica, mannan, steroid saponin phenanthrene.,,,,,,,,,,,,,.,,.,., Nigeria stew.,,,.,.,.,,,.,,,,,,,.,,,

27 glucomannan,,.,,..,,.,,,, ㆍ,, formulation

28 2 1..,,. 2007, 1~2mm ) AOAC 105., 2 g 105 dry oven ) (, 2005). 2 g , ) AOAC. (N) 2 3 mg 0.5 g

29 10 ml ) g, , 98~ ) 100,,,. 6) phenol-sulfuric acid method. Sample 1mL 10% phenol solution 1mL 10 H 2 SO 4 5mL nm. Glucose standard curve (%). 7) Sample 1mL 1mL 10 Boiling 1mL 520nm. Glucose stnadard curve (%). 8) (CR-300 series, Minolta, Japan) Hunter system L(, Lightness), a(, Redness)

30 b(, Yellowness). L=97.57, a=0.00, b= , 55, mesh... 1). erythritol, maltitol, sorbitol, xylitol(mitsubishi, Japan), mannitol(dae-jung, Korea), citric acid(sigma, USA), fumaric acid malic acid(dae-jung, Korea), tartaric acid(kanto, Japan), succinic acid(hayashi, Japan). 2~3 mm erythritol, maltitol, mannitol, sorbitol xylitol 10%, citric acid, fumaric acid, malic acid, succinic acid tartaric acid 0.2% 60 (HK-DO1000F, Korean Eng. Corp, Korea) 16, (18%)., (HMF-985, Hanil, Korea) mesh. 2), (CR-300 series, Minolta, Japan) Hunter system L(, Lightness), a (, Redness) b(, Yellowness). L=97.57, a=0.00, b=

31 . Schoch Kim., (%) 0.5 g 40 ml rpm ,000 g 30,., phenol-sulfuric acid, (g/g). Water solubility = Weight of total glucose in supernatant sample (g) Sample weight (g) 100 Swelling power = Weight of swollen sample (g) Sample weight (g) (100-%water solubility) 100 3) 30.. (brightness),,,. likert 9. 4) 3, statistical Analysis System(SAS) Duncan's multiple range test g homogenizer

32 100 ml shaking water bath (40, 50, 60, 70 ). %(v/w) ,000 g ). 100 ml 1%(w/w) 40, 50, 60, 70 3.,.,,,. 2) Table 1 1%(v/v), shaking water bath 3.,,,. 3) 0.05, 0.1, 0.5, 1.0, 2.0% (w/w).,,,. 4).,,,

33 Table 1. Temp ( ) Optimal ph Alcalase 55~70 6.5~8.5 Bacillus licheniformis,,, Neutrase 45~55 5.5~7.5 Bacillus amyloliquefaciens Flavourzyme ~7.0 Protamex 35~60 5.5~7.5 (N- ) Aspergillus oryzae Bacillus amyloliquefaciens, Bacillus licheniformis,,, (Table 2) 33 Enterococcus 5, Lactobacillus 14, Lactococcus 2, Leuconostoc 6, Pediococcus 2, Weissella 3 KCTC MRS (Difco, USA) mesh , 2%(v/v) 37 8, , 6,000 g, um syringe filter 1.8 ml eppepdorf tube ph,,,,,,

34 Table 2. No Strain Scientific name 1 KCTC 3638 Enterococcus casseliflavus 2 KCTC 3195 Enterococcus faecalis var. liquefaciens 3 KCTC 3552 Enterococcus flavescens 4 KCTC 3102 Enterococcus hirae 5 KCTC 3641 Enterococcus malodoratus 6 KCTC 3102 Enterococcus mundtii 7 KFRI 150 Lactobacillus acidophilus 8 KFRI 238 Lactobacillus amylophilus 9 KCTC 3102 Lactobacillus bervis 10 KFRI 1030 Lactobacillus bifermentans 11 KFRI 346 Lactobacillus casei 12 KFRI 1182 Lactobacillus collinoides 13 KFRI 654 Lactobacillus curvatus 14 KCTC 1047 Lactobacillus delbrueckii subsp. delbrueckii 15 KCTC 3112 Lactobacillus fermentum 16 KCTC 3602 Lactobacillus maltaromicus 17 KFRI 481 Lactobacillus pentosus 18 KCTC 1048 Lactobacillus plantarum 19 KCTC 3594 Lactobacillus reuteri 20 KCTC 3205 Lactobacillus sanfranciscensis 21 KFRI 684 Lactococcus lactis 22 KCTC 201 Lactococcus lactis subsp. cremoris 23 KCTC 3524 Leuconostoc carnosum 24 KCTC 3526 Leuconostoc citreum 25 KCTC 3102 Leuconostoc gelidum 26 KCTC 3528 Leuconostoc lactis 27 KCTC 3530 Leuconostoc mesenteroides subsp. dextranium 28 KCTC 3100 Leuconostoc mesenteroides subsp. mesenteroides 29 KFRI 832 Pediococcus pentosaceus 30 KCTC 3507 Pediococcus pentosacius 31 KCTC 3807 Weissella cibaria 32 KFRI 1184 Weissella confusa 33 KCTC 3531 Weissella paramesenteroides

35 4.. DPPH DPPH Blois (1958) DPPH(1,1-Diphenyl-2-picrylhydrazyl) radical radical 517 nm., 0.1 mm DPPH soln (99.8% Methanol), 517 nm 0.94~ mm DPPH soln (99.8% Methanol) 3 ml 1 ml nm. DPPH radical. scavenging effect (%) = [1 - Absorbance of sample517nm Absorbance of control517nm ] 100. (Fibrinolytic activity) Fibrin( ) Astrup. 50 mm (ph 7.4, 0.15M NaCl ) Fibrinogen 0.3% 5 ml 2% agarose ( ) 5 ml. thrombin(100nih unit/ml) 0.1 ml petri-dish 30~60 Fibrin plate. fibrin plate pasteur pipette 5 mm 7 20 ul 37 12, plasmin (1.0 unit/ml).. (%) = plasmin

36 . HMG-CoA (3-hydroxy-methylglutaryl coenzyme A) reductase HMG-CoA reductase Kleinsek (1989). 1 ml Cuvette 20 ul, 0.5 um ph ul, DTT(20 mm)100 ul, NADPH(3 mm) 100 ul, 100 ul 37 HMG-CoA(3 mm) 100 ul 340 nm 5. DMSO. (%) = 1 - (sample O.D -sample blank O.D) (control -control blank O.D) 100. (Bile acid binding capacity) Bile acid binding capacity Camire (1993)., 0.1 g 5 ml 0.1 N-HCl 2 ml N-NaOH ph 7.0 cholic acid, deoxycholic acid, glycocholic acid, taurocholic acid 31.25μ mol/ml 0.1 M phosphate buffer (ph 7.0) 4 ml porcine pancreatin 10 mg/ml 0.01 M phosphate buffer (ph 7.0) 5 ml M phosphoric acid 2 ml 26,890 g M phosphate buffer (ph 7.0) 5 ml vortex mixer. 1N-NaOH ph ml 3 ml test reagent(nitroamide dinucleotide, nitro blue tetrazolium salt, diaphorase, 3a-hydroxysteroid dehydrogenase) 0.5 ml. sample blank test reagent 3a-hydroxysteroid dehydrogenase. control control blank, control blank dehydrogenase, control 0.5 ml M phosphoric acid 0.1 ml 530 nm bile

37 acid. bile acid Fig. 1. Fig g 9, Fig. 2 1% 60 shaking water bath 90. 8,000 rpm, n-hexane, chloroform, ethyl acetate, n-butanol ethanol. 50 ml 100 ml, 20 ml 40 ml ml

38 . Fig. 2. Protamex. 100g 9 Lactobacillus acidophilus 1% ( cfu/g ) incubator 8000 rpm n-hexane, chloroform, n-butanol, ethyl acetate ethanol 5 separated funnel 40 ml 80 ml ml, 30 ml.. (ultrafiltration, UF), size-cut off

39 . 15 ml micro centrifuge tube membrane cell (ultrafree-15, Millipore) 3000 xg. 300,000 dalton, 300,000 dalton, 100,000~300,000 dalton, 100,000 dalton, 50,000~100,000 dalton, 50,000 dalton, 30,000~50,000 dalton, 30,000 dalton, 10,000~30,000 dalton, 10,000 dalton. Fig. 3.. gel chromatography (gel filtration chromatography). Sephedex G-75(3-80 kda). (φ cm), buffer(5 ml) loading (ph 7.0). fraction collector 6 ml.,,, HMG-CoA reductase, (Bile acid binding capacity)

40 . HMG-CoA reductase inhibition bile acid binding capacity, compound.. Gel chromatography ml 5% phenol 1 ml 5 ml nm.. Gel chromatography BCA Lowry Folin BCA. BCA(bicinchoninic acid) 4% CuSO 4 50:1, Standard Working Reagent(SWR). 1 well, SWR 142 ul 8 ul nm Calibration Curve, Curve., bovine serum albumin.. Gel chromatography Folch., 2 g chloroform/methanol(2:1, v/v) 20 ml 30 stirring (Whatman No.2). 20mL. Buchner funnel, round bottom flask rotary vacuum evaporator. Folch chloroform/methanol(2:1, v/v) 20 ml

41 Chloroform chloroform. Chloroform evaporator,.. Sepadex G-75 column. Sepadex G-75column(2.8 50cm) 100mM Tris-HCl buffer(ph 8.3) blue dextran void volume(vo). column elution volume(ve), Ve/Vo. Somatostatin(1.6 kda), Aprotinin(6.5 kda), Cytochrome C(12.4 kda), Carbonic Anhydrase(29 kda), Albumin(66 kda). * Total bed volume( ) ; column volume Elution volume( ) ; volume Void volume( ) ; volume Inner volume( ) ; volume ( : 30 cm). 500 ml 500 ml,

42 nm ) 3~6, 100~120 1 hr. 1~4, 1~2 (HMF-985, Hanil, Korea). 0.1~2%, 0.5~3%, 0.5~1% 25±2 24 hr.,,,, 25±2 10 days. 60~70 10~30 min

43 (, ) (100~120, 1~2hr) 1 (25±2, 24hr) 2 (25±2, 10 days) Fig ) (1).,,,

44 ,.,. 50%. 40~ Tempering 29 Table 3. ( cm) 4 35 min. Table 3. Ingredients Conc. 5% 10% 15% 20% 25% 30% 1) EH-D White chocolate Total ) EH-D: enzyme hydrolysate of Dioscorea batatas (2),,.. 40~42 25 Tempering 29 Table 4. ( cm) 4 35 min

45 Table 4. Conc. Ingredients 5% 10% 15% 20% 25% 30% 1) S-D White chocolate Total ) S-D: Steamed Dioscorea batatas (3). 40~42 25 Tempering 29 Table 5. ( cm) 4 35 min. Table 5. Conc. Ingredients 1% 3% 5% 7% 10% 1) P-D White chocolate Total ) P-D: Powder of Dioscorea batatas (4),.,,, 30 likert 9. (5)

46 50%. 10%, 15% 3%, 20% 20% 5%. 45~50 27 Tempering 32, ( cm) 15 min. 40~42 25 Tempering 29 Table 6. 10%, 15% 3% ( cm), 20% 20% 5% 2/3 1/ min. *tempering : tempering

47 Table 6. Ingredients Conc. 1) EH-D 2) S-D 3) P-D 10% 20% 15% 20% 3% 5% EH-D S-D P-D White chocolate Dark chocolate Total ) EH-D: enzyme hydrolysate of Dioscorea batatas 2) S-D: Steamed Dioscorea batatas 3) P-D: Powder of Dioscorea batatas 2) (1) 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%. 40% Table 7. (HMF-985, Hanil, Korea) 50 mesh 30 min,.. 30% 10 0 gel 10%,. ( cm)

48 Table 7. Ingredients 0.5% 1.0% 1.5% 2.0% 2.5% 3.0% Agar powder ) LF-D Sugar Oligosachharide Total 100 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas

49 (2) 30%, 35%, 40%, 45%, 50%. 10 %, Table 8. (HMF-985, Hanil, Korea) 50 mesh 30 min, gel 10%,. ( cm). Table 8. Ingredients 30% 35% 40% 45% 50% Agar powder ) LF-D Sugar Oligosachharide Total 100 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas (3) Table 9. (HMF-985, Hanil, Korea) 50 mesh 30 min, gel 10%,. ( cm)

50 (4) Table 9. (HMF-985, Hanil, Korea) 50 mesh 30 min, gel 10%,. ( cm). (5) Table 9. (HMF-985, Hanil, Korea) 50 mesh 30 min, gel 10%,. ( cm). Table 11., Ingredients Concentration (%) ) NF-D / 2) NP-D / 3) S-D Water Agar powder Granulated sugar Oligosaccharide Total 100 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas 2) NP-D: Not processed of Dioscorea batatas 3) S-D: Steamed Dioscorea batatas

51 (6) Table 12. (HMF-985, Hanil, Korea) 50 mesh 30 min, gel 10%,. ( cm). Table 12. Ingredients 3% 5% 7% P-D Water Agar powder Sugar Oligosaccharide Total 100 1) P-D: Powder of Dioscorea batatas (7), 10%, 20%, 30%, 5%,,,. 2%.,,, 48%( 14%, 8%, 8%, 8%, 10%), 2%. Table

52 10%, 20%, 30%, 5% (HMF-985, Hanil, Korea) 50 mesh. 2% 30 min, 100. gel 10%, ( cm). 50 mesh 30 min, 100 heating 10% ( cm) Fig

53 Table 13. Ingredients 5) WH-SJ 6) SK-SJ 7) CB-SJ 8) CS-SJ 9) PE-SJ 1) LF-D ) NP-D ) S-D ) P-D Water Agar Xanthan gum Sugar Oligosaccharide whey powder skim milk cocoa butter Citrus syrup plum extract Total 100 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas 2) NP-D: Not processed of Dioscorea batatas 3) S-D: Steamed Dioscorea batatas 4) P-D: Powder of Dioscorea batatas 5) WH-SJ, 6) SK-SJ, 7) CB-SJ, 8) CS-SJ and 9) PE-SJ means added with whey powder, skim milk, cocoa butter, citrus syrup, plum extract in sweet jelly, respectively. *Each sweet jelly included a Dioscorea batatas products( 1)-5) ) respectively.. 1) (1). (

54 70%, 30% ) 200 g 800 ml 60 2 hr. 10 Brix., 60 6 hr starter Lactobacillus acidophilus( CFU/mL) 1% hr.. Table 14. Ingredients Control Malt-B Sugar-B 1) LF-D Water Malt solution (10 Brix) Sugar % L. acidophilus Total ) LF-D: Lactic acid bacteria fermented by Dioscorea batatas (2). Lactobacillus acidophilus 1 2 Lactobacilus amylophilus Leuconostoc mesenteroides subsp. mesenteroides Lactobacilus amylophilus Lactobacillus acidophilus. Leuconostoc mesenteroides subsp. mesenteroides

55 , 2. Lactobacillus acidophilus( CFU/mL), Leuconostoc mesenteroids subsp. mesenteroids( CFU/mL) Lactobacillus amylophilus( CFU/mL) Table 14 1% hr. 50% 1 10% 1% ( CFU/mL) 2 L. acidophilus L. amylophilus L. amylophilus Leu. mesenteroides Leu. mesenteroides subsp. mesenteroides subsp. mesenteroides 37, 24hr L. acidophilus 37, hr Fig

56 (3) 10%, 20%, 30%, 40%, 50%. Leuconostoc mesenteroids subsp. mesenteroids( CFU/mL) Lactobacillus amylophilus( CFU/mL) 1% hr. Table % 20% 30% 40% 50% 1) LF-D Water Granulated sugar L. amylophilus Leu. mesenteroides subsp. mesenteroides Total ) LF-D: Lactic acid bacteria fermented by Dioscorea batatas (4),. 20%, 40%, 60%, 20%, 40%, 60%, 5%, 10%, 15%. Leuconostoc mesenteroids subsp. mesenteroids( CFU/mL) Lactobacillus amylophilus( CFU/mL) 1% hr. (5). 10%,

57 20%, 20%, 5%. 200 g, 200 g, 50 g, 100 g, 100 g 700 ml, 700 ml, 850 ml, 800 ml, 800 ml (HMF-985, Hanil, Korea) 50 mesh 900 ml. 900 ml,,,, vitamin C, L. acidophilus, L. amylohpilus, Leu. mesenteroides subsp. mesenteroides hr. 1) LF-D 10% 2) NP-D 20% 3) S-D 20% 4) P-D 5% X 5% 5% 0.2% vitamin C 0.1% 0.2% L. amylophilus 0.5% Leu. mesenteroides subsp. mesenteroides 0.5% 37, 24-48hr 1) LF-D: Lactic acid bacteria fermented by Dioscorea batatas 2) NP-D: Not processed of Dioscorea batatas 3) S-D: Steamed Dioscorea batatas 4) P-D: Powder of Dioscorea batatas Fig. 7. 2) (1)

58 . 5 %, 7 %, 10 %, 5 %, 10 %, 15 % Table %. Table 16. Ingredients 5% 7% 10% 1) LF-D Water Oligosachharide Rice powder Total ) LF-D: Lactic acid bacteria fermented by Dioscorea batatas (2)., 10 %, 20 %, 30 %. Table 17. Table 17. Ingredients 10% 15% 20% 1) NP-D Water Oligosaccharide Rice powder Total ) NP-D: Not processed Dioscorea batatas

59 (3).,,., Table 18. Table 18. Ingredients 20% 40% 60% 1) S-D Water Oligosaccharide Rice powder Total ) S-D: Steamed Dioscorea batatas (4).,.. Table

60 Table 19. Ingredients 3% 5% 7% 1) P-D Water Oligosaccharide Rice powder Total ) P-D: Powder of Dioscorea batatas (5) , (HMF-985, Hanil, Korea) 50 mesh. 150 g, 200g, 30 g, 50 g 700 ml, 700 ml, 820 ml, 800 ml (HMF-985, Hanil, Korea) 50 mesh. 40~50 80%. Table 20. Table 20. Ingredients Conc. 1) LF-D 5% 2) NP-D 15% 3) S-D 20% 4) P-D 3% Dioscorea batatas Water Oligosaccharide Rice powder Total ) LF-D: Lactic acid bacteria fermented by Dioscorea batatas 2) NP-D: Not processed of Dioscorea batatas 3) S-D: Steamed Dioscorea batatas 4) P-D: Powder of Dioscorea batatas

61 . 1) (intensity characteristics) (sensory characteristics). 30 Likert 9 1 " (extremely weak)", 9 " (extremely strong)", 1 " (extremely bad)", 9 " (extremely good)". 2) ph 10 ml ph meter (Metrohm 827). 3) 10 ml ph N NaOH lactic acid(%). 4) (PDX-1, Vee-gee corp,. U.S.A) 100 ul. 5) (CR-300 series, Minolta, Japan) Hunter system L(, Lightness), a(, Redness) b(, Yellowness). L=97.57, a=0.00, b= ) (MB45, Moisture analyzer, OHAUS Corp., Switzerland). 7) (DV- + RV viscometer, Brookfield, U.S.A)

62 8) 0.85% ph 5.5 MRS hr. Lactobacillus amylophilus starch agar (Difco, USA), Leuconostoc mesenteroides subsp. mesenteroides PES. Lactobacillus acidophilus 1, m-lbs starch.. 1),., (bloom). Bloom fat-bloom sugar-bloom, tempering.,, bloom, 10%, 15% bloom. bloom tempering tempering 30, 40, 50, 60 1 bloom. 2) probiotics,

63 . 5, 7.,, ,,.. 7. in-vivo. 1) 3 ICR mouse male group. group cholesterol pellet free feeding

64 Table 21. Group Diets Group I Group II cholesterol 2% Group III cholesterol 2% + 5% Group Ⅳ cholesterol 2% + LB. casei 5% Group Ⅴ cholesterol 2% + Lc. lactis 5% Group Ⅵ cholesterol 2% + En. flavescens 5% Group Ⅶ cholesterol 2% + Lb. acidophilus 5% Group Ⅷ cholesterol 2% + protamex 5% Group Ⅸ cholesterol 2% + Newtruse 5% Group Ⅹ cholesterol 2% + En. liqufaciens 5%. 1) 8 SD rat cage g (22 ) (60%) , cholesterol, lard pellet free feeding

65 Table 22. Group Diets GroupⅠ: NFD ( 4.3% ) GroupⅡ: HFD cholesterol 2% + 10% GroupⅢ: FEt-HFD cholesterol 2% + 10% + ethanol 10% GroupⅣ: FBu-HFD cholesterol 2% + 10% + butanol 10% GroupⅤ: PEA-HFD cholesterol 2% + 10% + protamex ethyl acetate 10% GroupⅥ: PEt-HFD cholesterol 2% + 10% + protamex ethanol 10%. 1) 4 SD rat (male) 10 9 group 90 7.,,, 22±2, 55±5%, 12 light-dark cycle. AIN-93G (Haraln Teklad, Madison, USA) Table ,

66 Table 23. Group Diets Items G1 - G2 ( + 10% lard + 2% cholesterol) - G3 + 10% - G4 + 10% G5 + 10% G6 + 10% G7 + base 10% - G8 + 10% - G9 + 10%. 1) Body weight group body weight g. 2) total Cholesterol total choleaterol kit., total cholesterol. blood serum total cholesterol. Cholesterol ester cholesterol cholesterol H 2 O 2. hydrogen peroxide peroxidase. Cholesterol ester > Cholesterol + fatty acids (by cholesterol esterase) cholesterol + O > cholest-4-en-3-ona + H 2 O 2 (by cholesterol oxidase) 2H 2 O AAP + p-hba------> Colored comp. +4H 2 O (by peroxidase) Serum 20 ul kit 3 ml

67 incubation spectrophotometer 500 nm, endpoint. 3) HDL-cholesterol cholesterol cholesterol ( ) cholesterol. cholesterol cholesterol. HDL (high density lipoprotein) cholesterol. HDL cholesterol. HDL (paraoxonase) LDL PAFAH(platelet activation factor acetylhydrolase). HDL cholesterol. Kit lipoprotein apo-lipoprotein B LDL (Low-Density lipoprotein) HDL (High-Density lipoprotein) cholesterol. HDL level. HDL-CHO assay kit 500 nm control. Serum 100 ul 100 ul 10 3,000 rpm ul 3 ml 37 5 spectrophotometer 500 nm, endpoint. 4) LDL-cholesterol LDL cholesterol total cholesterol, HDL cholesterol triglycerides friede-wald. LDL cholesterol= [total cholesterol-(hdl cholesterol + (triglyceride/5))]

68 5) Triglyceride serum complex lipoprotein. triglyceride determination triglyceride glycerol free fatty acid enzymatic akaline hydrolysis release glycerol. + L-a- H 2 O ESPT serum 20 ul 3 ml spectrophotometer 550 nm, endpoint. 6) GOT GPT GOT,,,,,,,. GPT GPT. GPT.. GPT. GPT., GOT.,. GOT GPT, GPT

69 GOT. Cholesterol vital response GOT, GPT level. GOT L-Aspartic acid + a-ketoglutaric acid >oxaloacetate + L-glutamate MDH Oxaloacetate + NADH + H >L-Malate + NAD+ + H2 GOT kit, GPT kit. 96-well plate sample 10 ul reagent R1+R2 (4:1) 100 ul, microplate reader (Molecular Device thermomax) 340 nm incubation 1 37 kinetic OD. 7) HMG-CoA reductase inhibition 1, 2 HMG-CoA reductase in-vitro. 3 HMG-CoA reductase in-vivo. HMG-CoA reductase Kleinsek. 300 ml KCl, 6 mm EDTA, 15 mm dithiothretol 240 mm potassium phosphate buffer 2 mm NADPH 100 ul, 1 mm HMG-CoA 100 ul 100 ul 37, 240 nm 6.22 mm/cm/l. 1 1 mg protein NADPH pmol. 8) 3 mean+standard deviation,

70 statistical Analysis System(SAS) Duncan's multiple range test

71 3 1..,,, Table 24., 5.66% 4.70%, 6.57% 5.09% % 9.88% 3%, 0.36% 0.15% % 79.23%. L( ) a( ) +1.60, +1.63, b( ) , % 0.43% 0.14% 0.10%.,

72 Table 24. Items ( ) ( ) Moisture (%) Ash (%) Crude protein (%) Crude lipid (%) Carbohydrate (%) L Color a b Total sugar content (%) Total Reducing sugar (%) Table 25, Table , %, % % % %, %

73 Table 25. Sample Dry Temperature ( ) Dry time (hr) , L , 60 L ( ), a( ) 2.03, b ( ) Table 26. Sample Dry Temperature ( ) Dry time (hr) L (Lightness) a (Redness) b (Yellowness) ), Table 27. mannitol erythritol Hunter-L (79.41),., tarta

74 ric acid Hunter-L Table 27. sugar alcohol organic acid Sample powder color value L(Lightness) a(redness) b(yellowness) Control 79.41± ± ±1.92 Erythritol 89.64± ± ±0.22 Mannitol 91.66± ± ±0.22 Maltitol 84.63± ± ±0.83 Sorbitol 83.05± ± ±0.43 Xylitol 83.52± ± ±0.55 Citric acid 86.80± ± ±0.28 Fumaric acid 86.48± ± ±0.65 Malic acid 85.70± ± ±0.37 Succinic acid 85.88± ± ±0.20 Tartaric acid 87.88± ± ±0.27, erythritol, tartaric acid,. erythritol maltitol. erythritol

75 Table 28. sugar alcohol organic acid Sample powder Water solubility (%) Swelling power (g/g) Control 5.94± ±0.02 Erythritol 10.37± ±0.06 Mannitol 6.61± ±0.08 Maltitol 5.61± ±0.06 Sorbitol 6.32± ±0.13 Xylitol 6.91± ±0.12 Citric acid 6.34± ±0.07 Fumaric acid 6.71± ±0.49 Malic acid 6.00± ±0.06 Succinic acid 6.53± ±0.03 Tartaric acid 6.75± ±0.13 2).., 10 : 1 (Table 29). Table 29. Ratio (water : powder) color viscosity taste overall acceptability 1 : ± ± ± ± : ± ± ± ± : ± ± ± ± : ± ± ± ± : ± ± ± ±1.26 Table 30. mannitol, maltitol, sorbitol erythritol 2. erythritol

76 erythritol. succinic acid, tartaric acid, malic acid. fumaric acid citric acid., ( ). Table 30. Sample powder Sugar alcohol Brightness Color preference Sweetness Sourness Taste preference Control 3.83±2.14 ab 4.67±1.37 a 5.33±1.97 c 3.50±2.07 c 5.83±1.83 bc Erythritol 6.00±2.83 b 5.00±2.83 a 8.00±1.79 a 2.17±1.17 c 7.67±1.63 abc Maltitol 7.33±1.97 ab 6.33±1.97 a 7.00±2.00 a 2.83±1.94 c 7.33±1.75 a Mannitol 7.75±0.50 b 5.25±0.50 a 7.75±0.96 a 1.50±0.58 c 6.75±2.06 abc Sorbitol 6.75±1.71 ab 5.75±0.96 a 5.25±1.71 c 2.50±3.00 c 5.75±2.22 bc Xylitol 3.17±1.47 a 4.33±2.34 a 7.50±2.35 ab 3.00±2.53 c 6.67±2.16 abc Citric acid 5.25±1.26 ab 4.25±2.06 a 2.75±1.50 c 5.00±2.16 a 5.50±0.58 c Fumaric acid 4.67±2.58 ab 5.00±2.10 a 3.00±1.10 c 4.50±1.76 bc 7.33±2.42 bc Organic acid Malic acid 6.17±1.17 ab 5.00±2.53 a 2.83±1.33 c 5.33±2.42 bc 5.17±0.98 c Succinic acid 6.25±1.71 b 5.00±1.83 a 1.00±0.00 c 7.00±0.82 ab 4.25±0.50 c Tartaric acid 6.75±2.36 ab 4.50±2.65 a 2.25±2.50 c 7.00±0.82 a 5.25±0.96 c * Superscript letters indicate significant difference at α=0.05 as determined by Duncan's multiplerange test. 2.. ( ) (AMG, Viscozyme, Pectinex, Celluclast 1.0%(w/w)) 40, 50, 60, 70., (Table 31) (Table 32) Celluclast 2~4-75 -

77 ., AMG Viscozyme, Pectinex, Celluclast. (Table 33) 0.5% Celluclast 1.0~3.6% 2, 50, 60,., AMG. (Table 34) 0.15% 0.3~1.34% 2,. AMG, 60. (Table 35) 60, AMG Viscozyme, Pectinex, Celluclast 40, 60 20%. AMG 30%. 60. Table 31. ( Brix) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before

78 Table 32. ( Brix) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before Table 33. ( Brix) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before Table 34. ( Brix) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before

79 Table 35. (%) Hydrolysis temp Carbohydrate enzyme AMG Viscozyme Pectinex Celluclast before ( ) (Alcalase, Neutrase, Flavourzyme, Protamex 1.0%(w/w)) 40, 50, 60, 70., (Table 31) (Table 32) Alcalase 2~4., Protamex Neutrase, Flavourzyme, Alcalase. (Table 33) 0.5% Alcalase 1.0~3.6% 2, 50, 60,., Protamex. (Table 34) 0.15% 0.3~1.34% 2,. Protamex, 60. (Table 35) 60, Protamex Neutrase, Flavourzyme, Alcalase 40, 60 20%. Protamex 30%

80 . 60. Table 31. ( Brix) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before Table 32. ( Brix) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before Table 33. ( Brix) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before

81 Table 34. ( Brix) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before Table 35. (%) Hydrolysis temp Protein enzyme Protamex Neutrase Flavourzyme Alcalase before ~2.0%(w/w) 90. (Table 36) (Table 37), Alcalase 1.5. Protamex, 0.5%. Neutrase 0.5%., Neutrase. (Table 38) Alcalase %

82 . Protamex, 0.5% 2.8g/100g Neutrase, Flavourzyme 3., Neutrase 0.5%., Neutrase. (Table. 39), 0.5%. Protamex, 0.5% 1.6g/100g 4, Neutrase, Flavourzyme, Alcalase. (Table 40) 0.05%. Protamex 0.5%. Neutrase 0.5% 2%.,,. Table 36. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before

83 Table 37. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before Table 38. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before Table 39. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before

84 Table 40. Hydrolysis Conc. Protein enzyme Protamex Neutrase Flavourzyme Alcalase before ~240., (Table 41) (Table 42). 60~ (Table 43). Protamex 60 Neutrase, Flavourzyme, Alcalase Neutrase Flavourzyme, Alcalase 60. (Table 44) 3. (Table 45)

85 60 50%. Table 41. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before

86 Table 42. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before Table 43. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before

87 Table 44. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before Table 45. Hydrolysis time Protein enzyme (min) Protamex Neutrase Flavourzyme Alcalase before ph,, Table ph ph ph 4.2~5.5, 2 ph. (Table 47) 0.5% 2 0.1~0.5%, 2. (Fig. 8) (Fig

88 9) 10 6 ~10 7 CFU/mL CFU/mL, CFU/mL 2. KFRI 150 (Lactobacillus acidophilus) 2., 2 ph 2 ph.., (Table 48) 1.5 Brix 2 2.0~6.0 Brix. KCTC 3102 (Enterococcus mundtii) 5.7~6.3 Brix KCTC 3195 (Enterococcus faecalis var. liquefaciens), KCTC 3102 (Lactobacillus bervis), KFRI 832 (Pediococcus pentosaceus) Brix 4 4.0~5.3 Brix. KCTC 3524 (Leuconostoc carnosum), KCTC 3205 (Lactobacillus sanfranciscensis), KCTC 201 (Lactococcus lactis subsp. cremoris), KCTC 3112 (Lactobacillus fermentum), KFRI 238 (Lactobacillus amylophilus), KFRI 150 (Lactobacillus acidophilus) 2.4~3.7 Brix Brix. (Table 49) 10.0% ~206.1% 2. KCTC 3638 (Enterococcus casseliflavus), KCTC 1048 (Lactobacillus plantarum ) 8.06, 8.95 g/100g , g/100g. KCTC 3102 (Lactobacillus bervis), KFRI 238 (Lactobacillus amylophilus), KFRI 481 (Lactobacillus pento ), KCTC 3594 (Lactobacillus reuteri), KCTC 201 (Lactococcus lactis subsp. cremoris) 7.2~9.8 g/100g ~

89 g/100g ~70.1 g/100g. (Table 50) 3.0 g/100g. KFRI 238 (Lactobacillus amylophilus) 3.48 g/100g 5, g/100g. KFRI 481 (Lactobacillus pentosus), KFRI 684 (Lactococcus lactis), KCTC 3507 (Pediococcus pentosacius) 3.5 g/100g g/100g

90 Table 46. ph Strain Fermentation time (days) KCTC KCTC KCTC KCTC KCTC KCTC KFRI KFRI KCTC KFRI KFRI KFRI KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KCTC KCTC KCTC KCTC KFRI KCTC KCTC KFRI KCTC

91 Table 47. Strain Fermentation time (days) KCTC KCTC KCTC KCTC KCTC KCTC KFRI KFRI KCTC KFRI KFRI KFRI KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KCTC KCTC KCTC KCTC KFRI KCTC KCTC KFRI KCTC

92 Fig

93 Fig

94 Table 48. Strain Fermentation time (days) KCTC KCTC KCTC KCTC KCTC KCTC KFRI KFRI KCTC KFRI KFRI KFRI KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KCTC KCTC KCTC KCTC KFRI KCTC KCTC KFRI KCTC

95 Table 49. Strain Fermentation time (days) KCTC KCTC KCTC KCTC KCTC KCTC KFRI KFRI KCTC KFRI KFRI KFRI KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KCTC KCTC KCTC KCTC KFRI KCTC KCTC KFRI KCTC

96 Table 50. Strain Fermentation time (days) KCTC KCTC KCTC KCTC KCTC KCTC KFRI KFRI KCTC KFRI KFRI KFRI KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KCTC KCTC KCTC KCTC KFRI KCTC KCTC KFRI KCTC

97 4.. HMG-CoA reductase Table 51 HMG-CoA reductase. 50, 0.5%, 90,,,,, HMG-CoA reductase. 1.6 cm. Table 51. HMG-CoA reductase Fibrinolytic activity (cm) HMG-CoA reductase (%) Control Alcalase Flavourzyme Protamex Neutrase HMG-CoA reductase Protamex %, Alcalase 45.32%, Neutrase 32.90% Flavourzyme. control HMG-CoA reductase.. (Bile acid binding capacity) 41% cholate, 39% chenodeoxycholat, 15% deoxycholate, 4% ursodeoxycholate, 1% lithocholate

98 cholic acid sodium salt glycine taurine glycoholic acid sodium salt taurocholic acid sodium salt, 1 7-a-dehydroxylation 2 2 deoxycholic acid sodium salt., ( 1%). feedback,. Table 52.. cholic acid 5.89% deoxycholic acid, taurocholic acid, glycocholic acid. Protamex glycocholic acid 15.91%, deoxycholic acid 12.07%. Neutrase glycocholic acid 9.63% Protamex glycocholic acid., protamex 4 glycocholic acid deoxycholic acid. Table 52. Enzyme Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid Control Alcalase Flavourzyme Protamex Neutrase

99 . Fibrinolytic activity (Table 53), KFRI 150 (Lactobacillus acidophilus), KCTC 3552 (Enterococcus flavescens), KCTC 3100 (Leuconostoc mesenteroides subsp. mesenteroides) 3, 2, 4. thrombin fibrinogen fibrin. (fibrin clots) plasmin (fibrinolytic enzyme), (thrombosis)., 33.. HMG-CoA reductase Table 54 HMG-CoA reductase. KCTC 3552 (Enterococcus flavescens) % KCTC 3195 (Enterococcus faecalis var. liquefaciens) % KCTC 3552 (Enterococcus flavescens). KFRI 150 (Lactobacillus acidophilus) %, KCTC 3112 (Lactobacillus fermentum) %, KFRI 654 (Lactobacillus curvatus) 90.1% KCTC 3526 (Leuconostoc citreum) 59.17% HMG-CoA reductase

100 Table KFRI 150 (Lactobacillus acidophilus) 4 cholic acid 8.12%, deoxycholic acid 9.80%, taurocholic acid 7.60%, glycocholic acid 6.98%. KCTC 3507 (Pediococcus pentosacius) cholic acid 9.58%, deoxycholic acid 8.02%, taurocholic acid 7.41%, glycocholic acid 6.30%

101 Table 53. Strain Fermentation time (days) KCTC KCTC KCTC KCTC KCTC KCTC KFRI KFRI KCTC KFRI KFRI KFRI KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KCTC KCTC KCTC KCTC KFRI KCTC KCTC KFRI KCTC

102 Table 54. HMG-CoA reductase Strain Fermentation time (days) KCTC KCTC KCTC KCTC KCTC KCTC KFRI KFRI KCTC KFRI KFRI KFRI KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KCTC KCTC KCTC KCTC KFRI KCTC KCTC KFRI KCTC

103 Table 55. Fermentation time (days) Strain C D T G C D T G C D T G Control KCTC KCTC KCTC KCTC KCTC KCTC KFRI KFRI KCTC KFRI KFRI KCTC KCTC KCTC KFRI KCTC KCTC KCTC KFRI KFRI KCTC KFRI KCTC KCTC KCTC KCTC KCTC KCTC KFRI KCTC KCTC KFRI KCTC C : Cholic acid, D : Deoxycholic acid, T : Taurocholic acid, G : Glycocholic acid

104 5.. protamex HMG-CoA reductase, bile acid, DPPH. HMG-CoA reductase Table 56.. Bile acid glycocholic acid. plate DPPH 43.2% 2. Table 56. HMG-CoA reductase Sample Inhibition activity (%) Supernatant 84.5 Precipitate 75.1 Table 57. Binding capacity (um) Sample Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid Supernatant Precipitate Table 58. Sample Fibrinolytic activity (cm) Supernatant - Precipitate

105 Table 59. DPPH Sample DPPH radical scavenging effect (%) Supernatant 43.2 Precipitate ) HMG-CoA reductase Table 60. ethanol 76.5%. 10~30%. Table 60. HMG-CoA reductase Item Solvent Inhibition activity (%) n-hexane 31.0 CHCl 상-상 1) E.A 51.9 n-buoh 36.4 EtOH 76.5 n-hexane 13.9 CHCl 상-침 2) E.A 21.2 n-buoh 13.1 EtOH 20.7 n-hexane 12.7 CHCl 침-상 3) E.A 18.8 n-buoh 15.2 EtOH 33.2 n-hexane 5.3 CHCl 침-침 4) E.A 10.6 n-buoh 4.5 EtOH 5.9 1) 2) 3) 4)

106 2) Bile acid binding capacity Table 61.. ethanol ethyl acetate 4 deoxycholic acid glycocholic acid. Table 61. Item 상-상 1) 상-침 2) 침-상 3) 침-침 4) Binding capacity (um) Solvent Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid n-hexane CHCl E.A n-buoh EtOH n-hexane CHCl E.A n-buoh EtOH n-hexane CHCl E.A n-buoh EtOH n-hexane CHCl E.A n-buoh EtOH ) 2) 3) 4)

107 3) Table 62.. Table 62. 상-상 1) 상-침 2) 침-상 3) 침-침 4) Sample Fibrinolytic activity (cm) n-hexane - CHCl 3 - E.A - n-buoh - EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - 1) 2) 3) 4) 4) Ethyl acetate 69.2%, Ethanol 50.9% n-butanol 45.1%

108 Table 63. DPPH 상-상 1) 상-침 2) 침-상 3) 침-침 4) Sample DPPH radical scavenging effect (%) n-hexane - CHCl E.A 69.2 n-buoh 45.1 EtOH 50.9 n-hexane - CHCl 3 - E.A 4.5 n-buoh - EtOH 6.7 n-hexane - CHCl 3 - E.A - n-buoh 16.9 EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - 1) 2) 3) 4)

109 . 1) HMG-CoA reductase, bile acid, DPPH.. HMG-CoA reductase 72.3% bileacid 4,. 65.1%. Table 64. HMG-CoA reductase Sample Inhibition activity (%) Supernatant 72.3 Precipitate 9.7 Table 65. Sample Binding capacity (um) Cholic acid Deoxycholic acid Taurocholic acid Glycocholic acid Supernatant Precipitate Table 66. Sample Fibrinolytic activity (cm) Supernatant - Precipitate - Table 67. DPPH Sample DPPH radical scavenging effect (%) Supernatant 65.1 Precipitate

110 . 1) HMG-CoA reductase HMG-CoA reductase inhibition. Table 68. HMG-CoA reductase ethanol (95.2%). Table 68. HMG-CoA reductase Item Solvent Inhibition activity (%) 상 - 상 1) 상 - 침 2) n-hexane 46.6 CHCl E.A 87.9 n-buoh 84.0 EtOH 95.2 n-hexane 39.8 CHCl E.A 40.9 n-buoh 39.8 EtOH ) 2) 2) Bile acid binding capacity Table 69. cholic acid, deoxycholic acid, taurocholic acid glycocholic acid n-butanol Ethanol. n-hexane

111 Table 69. Item Solvent Cholic acid Binding capacity (um) Deoxycholic acid Taurocholic acid Glycocholic acid n-hexane CHCl 상 - 상 1) E.A n-buoh EtOH n-hexane 상 - 침 2) CHCl E.A n-buoh EtOH ) 2) 3) Table 70.. Table 70. Sample Fibrinolytic activity (cm) 상 - 상 1) 상 - 침 1) n-hexane - CHCl 3 - E.A - n-buoh - EtOH - n-hexane - CHCl 3 - E.A - n-buoh - EtOH - 1) 2)

112 4) Table 71. Ethanol 68.9%. n-butanol 53.3%. n-hexane 4.2%. n-hexane chloroform, Ethanol 9.1%. Table 71. DPPH Sample DPPH radical scavenging effect (%) 상 - 상 1) 상 - 침 2) n-hexane 4.2 CHCl E.A 34.4 n-buoh 53.3 EtOH 68.9 n-hexane - CHCl 3 - E.A 5.2 n-buoh 3.6 EtOH 9.1 1) 2). 1) HMG-CoA reductase ethanol HMG-CoA table 72. HMG-CoA reductase 93.4%. 100,

113 5.7~18.9% 30,000~ 50, %. ethanol 30,000~50, %. 30, % 30,000. Table 72. ethanol HMG-CoA reductase Inhibition activity (%) (dalton) Control* , , , , , , , , ,000-50, , ,000-30, , Control*: ethanol 2) ethanol HMG-CoA reductase 30,000~50, deoxycholic acid 30,

114 . HMG-CoA reductase ethanol 30,000~50,000 gel-filtration. Table 73. ethanol bile acid binding capacity Cholic acid Binding capacity (um) Deoxycholic acid Taurocholic acid Glycocholic acid Control* , , , , , , , , ,000-50, , ,000-30, , Control*: ethanol

115 Table 74. ethanol bile acid binding capacity Cholic acid Binding capacity (um) Deoxycholic acid Taurocholic acid Glycocholic acid Control* , , , , , , , , ,000-50, , ,000-30, , Control*: ethanol. Gel chromatography 1) Gel chromatograpy Gel filtration 280 nm O D (Fig. 10, Fig. 11). 50,000~30,000 dalton gel filtration 280 nm Fig ~30 40~50 peak. 30,000 dalton gel filtration Fig (Ⅰ : 10~45, Ⅱ : 51~60, Ⅲ : 86~100, Ⅳ : 121~125)

116 Fig. 10. Gel chromatograpy Sephedex G-75 (280 nm) * : 탄수화물, 지질, 단백질측정 fraction

117 Fig. 11. Gel chromatograpy Sephedex G-75 (280 nm) * : 탄수화물, 지질, 단백질측정 fraction 2) fraction No. 11~30 41~50, fraction No. 16~ %. (Ⅰ : 11~45, Ⅱ : 51~60, Ⅲ : 86~100, Ⅳ : 121~125) fraction, Ⅰ 34~40 15%, Ⅱ 52~58 22% Ⅲ Ⅳ

118 Table 75. Gel chromatograpy Fraction number Total carbohydrate (%) Ⅰ Ⅱ

119 Table 76. Gel chromatograpy Fraction number Total carbohydrate (%) Ⅰ continued

120 Ⅱ Ⅲ Ⅳ ) gel filtration, Ⅰ fraction No. 26~ ~13.2%, Ⅱ 45 11%.. Ⅰ 10% Ⅱ 54~56 15%. Ⅲ Ⅳ % 17.2%

121 Table 77. Gel chromatograpy Fraction number Total protein (%) Ⅰ Ⅱ

122 Table. 78. Gel chromatograpy Fraction number Total protein (%) Ⅰ continued

123 Ⅱ Ⅲ Ⅳ ), Ⅰ %. fraction, Ⅰ fraction No.36~ ~13.2%, Ⅱ Ⅲ

124 Table. 79. Gel chromatograpy Fraction number Total lipid (%) Ⅰ Ⅱ

125 Table 80. Gel chromatograpy Fraction number Total protein (%) Ⅰ continued

126 Ⅱ Ⅲ Ⅳ ,000~50,000 dalton 30,000 dalton gel filtration 280 nm 2, 4., Ⅰ 21~25 45%, 13%, 5%. Ⅱ Ⅰ 45 9%, 11%

127 1%. Ⅰ 35~40 15%, 8%, 13.2% Ⅱ 55 23%, 16%. 5%. Ⅲ 95 10%. Ⅳ Ⅲ 17%. Ⅰ Ⅱ Ⅰ,, Ⅱ. Ⅲ Ⅳ. 5) HMG-CoA reductase,, peak fraction HMG-CoA reductase., 10% HMG-CoA reductase, Ⅰ fraction No. 21~22 52%. 4, HMG-CoA reductase, Ⅰ Ⅳ 10~20%. Ⅱ % Ⅲ %

128 Table 81. Gel chromatograpy HMG-CoA reductase Fraction number Inhibition activity (%) Table 82. Gel chromatograpy HMG-CoA reductase Fraction number Inhibition activity (%)

129 6) 2 HMG-CoA reductase fraction No Taurocholic acid. 4 HMG-CoA reductase Ⅰ, Ⅳ 5 um. Ⅱ 55~57 Ⅲ 93~95. Table 83. Gel chromatograpy Fraction Binding capacity (um) number Cholic acid Deoxycholic Taurocholic Glycocholic acid acid acid 분획 분획

130 Table 84. Gel chromatograpy Binding capacity (um) Fraction number Deoxycholic Taurocholic Glycocholic Cholic acid acid acid acid 분획 분획 분획 분획 ) (Sephadex G-75, ((3-80 kda)). Fig nm. gel filtration chromatography molecular weight gel column molecular weight gel column. volume (Table 85). molecular weight Fig. 12 Albumin(66 kda), Carbonic Anhydrase(29 kda), Cytochrome C(12.4 kda), Aprotinin S(6.5 kda), Aomatostatin(1.6 kda)

131 Table 85. Gel filtration Sample Molecular weight (Da) Log molecular weight (ml) Somatostatin Aprotinin Cytochrome C Carbonic Anhydrase Albumin * π 길이 r=2.8 cm, =50 cm number Fracration Fig. 12. Sephadex G-75 gel filtration peak

132 Albumin ES-2 Carbonic anhydrase Cytochrome C Aprotinin ES Somatostatin number Fraction Fig. 13. molecular weight * ES-2: enzyme hydrolysis sample from Dioscorea batatas (no. 56) * ES-3: enzyme hydrolysis sample from Dioscorea batatas (no. 95) 4 Ⅱ Ⅲ. Ⅱ 56, Ⅲ 95 HMG-CoA reductase peak kda Carbonic Anhydrase, kda Somatostatin

133 Albumin FS Carbonic anhydrase Cytochrome C Aprotinin 5000 Somatostatin number Fraction Fig. 14. molecular weight * FS-1: fermented sample from Dioscorea batatas (no. 22) M.W (Fig. 14). Ⅰ HMG-CoA reductase Ⅱ 22. Fig. 12 peak kda albumin.. 1) ethyl acetate ethanol. maillard reaction

134 Fig. 15. Fig. 16. protamex

135 . 100,000 dalton 50,000 dalton, 30,000 dalton, 10,000 dalton. Fig. 17. Fig

136 2) n-butanol ethanol, ethyl acetate. maillard reaction. Fig. 19. Fig

137 , 100,000 dalton.. Fig. 21. Fig

138 6.. 1) ph 5 ph, 100:0, 80:20, 50:50, 20:80 ph 6.2~6.5, ph 4.3, 0:100 ph 6.89, ph Table 86. ph : 100:0 80:20 50:50 20:80 0: ) 5, 100:0, 80:20, 50:50, 20:80, 0:

139 Fig ),..,.,. 20:

140 Table 87. : 100:0 80:20 50:50 20:80 0: ) ph,, ph 4.. ph 4.3, 0.33, 15%. Table 88. ph,, : 0:0 0:1 1:0 1:1 ph (%) (%) ),,,,

141 Table 89. : 0:0 0:1 1:0 1: ) Table 90, 5 %, 20 %, 10 %. Table 90. 5% 10% 15% 20% 25% 30% ( ) ) Table 91,.,, 15%

142 Table 91. 5% 10% 15% 20% 25% 30% ( ) ) Table 92,. 3%. Table 92. 1% 3% 5% 7% 10% ( )

143 Fig ), 2%. 1.5%, 2.5%. Table % % % % % % )

144 , 50%,. Table % % % % % ), 10% 20% 30%. Table % % % ) 20%, 30%

145 Table % % % ) 30%. Table % % % ) 5%,. Table 98. 3% % % ),,,,,,,

146 %, 50 % 10 %, 20 %, 5 %.,,. Table % 20% 30% 5% Fig